The invention is particularly useful in the field of health care as well as research in biological and medical science, particularly in nucleic acid analysis, gene quantification and genotyping, where reliable analysis of samples for components contained therein is needed. Monitoring chemical reactions by the use of optical systems is well known, for example in molecular diagnostics, where chemical reactions involving nucleic acids are detected and quantified by fluorescent dyes (e.g. propidium iodide, cybergreen, acridine orange) that intercalate between the stacked bases at the centre of the DNA double helix or where specific products of the chemical reaction are detected and quantified by oligonucleotides labeled with compounds such as fluorescein, rhodamine or cyanine dyes where these labeled oligonucleotides specifically hybridize to target DNA sequences. An important aspect of such chemical reactions is monitoring the reaction by exciting the dyes via beams of light of specific wavelengths and measuring the light emitted by these dyes. Precision in these steps is a prerequisite for the accuracy of such methods.
The polymerase chain reaction (PCR) has revolutionized the field of nucleic acid treatment, particularly the analysis of nucleic acids, by providing a tool to increase the amount of nucleic acids of a particular sequence from negligible to detectable amounts. PCR is described e.g. in EP 0201184 and EP 0200362. An instrument for performing PCR in a controlled manner on samples in tubes by heating and cooling an extended metal block is disclosed e.g. in EP 0236069.
More recently improved and more powerful PCR techniques have been developed. Quantitative real time PCR is a technique used to simultaneously amplify and quantify a specific part of a given DNA molecule. It is used to determine whether or not a specific sequence is present in the sample and if present, the number of copies in the sample can be quantified. Two common methods of quantification are the use of fluorescent dyes that intercalate with double-strand DNA and modified DNA oligonucleotide probes that fluoresce when hybridized with a complementary DNA.
Furthermore, multiplex PCR techniques which enable amplification of two or more products in parallel in a single reaction tube, have been developed. These techniques may be widely used in genotyping applications and different areas of DNA testing in research, forensic, and diagnostic laboratories. Multiplex PCR can also be used for qualitative and semi-quantitative gene expression analysis using cDNA originating from a variety of eukaryotic and prokaryotic sources as a starting template.
Various instruments for performing, detecting, and monitoring such methods are known in the art. The Roche Cohas® TaqMan® instrument (see e.g., EP 0953837) and the Roche Lightcycler® 480 instrument make use of a white light source for providing excitation beams to a sample. Using a conventional white light source as an excitation light source is disadvantageous, as the lifespan of such white light sources generally is below 1000 hours of operation leading to increased maintenance efforts and costs. Furthermore, the spectral power of some white light sources (particularly halogen bulbs) is rather low in the desirable blue range, having only limited energy for exciting the sample and leading to elongated measuring times. Furthermore, white light sources generate a broad spectrum of spectral wavelengths, necessitating the use of expensive filters because a vast fraction of generated light of other wavelengths is not used in the application and needs to be blocked. In addition, white light sources produce heat which needs to be conducted away from the instrument.
Some instruments known in the art produce excitation beams by a single light-emitting diode (e.g., the Roche Lightcycler® 1.5 and 2.0) or by a laser (e.g., the ABI Prism 7700, 7900 as described in WO 2003/098278). Other instruments employ multiple light-emitting diodes of the same wavelength (e.g., the Eppendorf Mastercycler® realplex instrument described in WO 2003/002991). These instruments are necessarily limited to a single excitation wavelength. Consequently hydrolysis multiplex applications can not be performed without complex modifications in the instrument because the set of useful dyes is limited by the excitation wavelength.
Still other instruments such as the Cepheid Smartcycler (see U.S. Pat. No. 6,369,893), use several light-emitting diodes of different wavelengths for exciting the chemical reaction being tested. Several detectors are used for detection of the emitted light from the same reaction. This is disadvantageous if a plurality of reactions need to be analyzed because all the components of the instrument, such as LEDs and detectors, may need to be multiplied by the number of reactions to be analyzed. In addition, the number of filters and dichroic mirrors, as well as the electronic circuits for driving the LEDs and preamplify the signals of the photodiodes may also need to be increased. In addition, the complexity is still more increased when more than 4 LED types of distinct wavelengths are used. Therefore, when several reactions need to be detected with a detection system such as described in U.S. Pat. No. 6,369,893, the complexity and costs of the detection system become very high.
Also known in the art are spectrometer instruments, such as those disclosed in DE 4424961. Such instruments use a static light source and optical fibers, each fiber being connected on the one end to one sample area and on the other end to a rotary wheel. By turning the wheel each reaction region of the sample area may be brought into optical contact with the static light source. However, an instrument with this concept has the disadvantage that the light of the single light source is applied sequentially to each reaction region. It is not possible to detect several dye-markers with distinct excitation and emission spectra in several reaction regions in parallel with this instrument. This results in long measurement times, especially when a lot of samples need to be measured repeatedly, leading to long overall process times.